Synthesis and antiviral evaluation of bis(POM) prodrugs of (E)-[4′-phosphono-but-2′-en-1′-yl]purine nucleosides

Article abstract of DOI:10.1016/j.ejmech.2012.08.042

Seventeen hitherto unknown bis(POM) prodrugs of novel (E)-[4′- phosphono-but-2′-en-1′-yl]purine nucleosides were prepared in a straight approach and at good yields. Those compounds were synthesized by the reaction of purine nucleobases directly with the p

Full text of DOI:10.1016/j.ejmech.2012.08.042

European Journal of Medicinal Chemistry 57 (2012) 126e133
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiviral evaluation of bis(POM) prodrugs of (E)-[40-phosphono-
but-20-en-10-yl]purine nucleosides
Ugo Pradère a, Vincent Roy a, Aurélien Montagu a, Ozkan Sari a, Manabu Hamada a, Jan Balzarini b,
,
Robert Snoeck b, Graciela Andrei b, Luigi A. Agrofoglio a
*
a Institut de Chimie Organique et Analytique, UMR 7311 CNRS, Université d’Orléans, 45067 Orléans, France
b Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Seventeen hitherto unknown bis(POM) prodrugs of novel (E)-[40-phosphono-but-20-en-10-yl]purine
nucleosides were prepared in a straight approach and at good yields. Those compounds were synthe-
sized by the reaction of purine nucleobases directly with the phosphonate synthon 3 bearing POM
biolabile groups under Mitsunobu conditions. All obtained compounds were evaluated for their antiviral
activities against a large number of DNA and RNA viruses including herpes simplex viruses 1 and 2,
varicella zoster virus, Feline herpes virus, human cytomegalovirus, HIV-1 and HIV-2. Among these
molecules, some of them exhibit anti-VZV and anti-HIV activity at submicromolar concentrations. This
class of compound will be of further interest for lead optimization as anti-infectious agents.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Article history:
Received 27 April 2012
Received in revised form
28 August 2012
Accepted 29 August 2012
Available online 7 September 2012
Keywords:
Acyclic nucleoside phosphonate
Prodrugs
Mitsunobu reaction
Antiviral evaluation
1. Introduction
phosphonate diester(s) with decreased polarity. Based on that,
PMEA and PMPA have been commercialized as their prodrug bis(-
Acyclonucleoside phosphonates (ANPs) [1] represent a key class
of antiviral drugs which has attracted considerable attention
through the large number of modified nucleobase and/or acyclic
chain moieties. Among ANPs, 9-[2-(phosphonomethoxy)ethyl]
adenine (PMEA) [2] or (R)-[2-(phosphonomethoxy)propyl]adenine
(PMPA) [3] exhibited strong antiviral activities against of HIV and/
or HBV infections (Fig. 1). However, their antiviral efficiency is
mainly hampered by their low cell penetration (<5%) but also by
their ability to be converted by human/viral kinases to their
triphosphate active forms at a high concentration at the right
location [4]. To circumvent the poor bioavailability of ANP which
are negatively charged at physiological pH, several groups [5e8]
have successfully developed biolabile promoieties in which the
charged phosphonic acid group is transformed into a neutral
POM)PMEA (Adefovir, HEPSERAÔ) and bis(POC)PMPA (Tenofovir,
VIREADÔ), respectively.
Part of our program to discover antiviral compounds, we have
recently reported a new class of acyclic nucleoside phosphonates
bearing the (E)-40-phosphono-but-20-en-10-yl side chain moiety,
which can mimic the conformation of the C1eO4eC4eC5 atoms
from the 2-deoxyribose in dTMP [9,10]. Having in hand an acyclic
phosphonate side chain, it was rational to connect this synthon to
purine base analogues which exhibit the greatest activities as
antiviral agents. In this work, we aim to identify a novel series of
purine ANP analogues in their prodrug form, combining our bio-
labile phosphonate side-chain and purine analogues selected in the
literature as the lead nucleobases which possesses antiviral prop-
erties (e.g., guanine [11] and its tricyclic analogues [12,13], N6-
substituted-purines [14,15]). Herein we report the biological eval-
uation and the efficient one step synthesis via Mitsunobu reaction
of (E)-bis-(POM)-[4-phosphono-but-20-en-10-yl]purine analogues.
Abbreviations: VZV, varicella zoster virus; VV, vaccinia virus; HSV, herpes
simplex virus; VSV, vesicular stomatitis virus; DNA, deoxyribonucleic acid; RNA,
ribonucleic acid; CC50, compound concentration affording 50% inhibition of cell
growth; EC50, compound concentration affording 50% inhibition of the viral cyto-
pathicity; MCC, minimum cytotoxic concentration required to afford a microscopi-
cally detectable alteration of cell morphology; MDCK, MadineDarby canine kidney.
* Corresponding author. Tel.: þ33 2 3849 4582.
2. Chemistry
We have previously reported the use of cross metathesis
reaction between bis(POM)-allyl phosphonate 2 and crotylated
E-mail address: luigi.agrofoglio@univ-orleans.fr (L.A. Agrofoglio).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.042

U. Pradère et al. / European Journal of Medicinal Chemistry 57 (2012) 126e133
127
The resulted mixture of two isomers (E and Z) was separable by
column chromatography to afford the desired isomer trans 3 in 74%
yield. A previous attempted cross metathesis reaction with croty-
lalcohol afforded also (E/Z) mixture in a disappointing 32% yield,
which can be mainly ascribed to the formation of the unreactive
crotyl phosphonate.
The synthesis of purine ANP analogues 9e13 was performed
as shown in Scheme 3). The key intermediates 7 and 8 were
obtained in 71% and 50% yield, respectively, through Mitsunobu
reaction between appropriate 6-chloropurines (5 or 6) and syn-
thon 3 in the presence of PPh3 and DIAD in dioxane at room
temperature. The Mitsunobu reaction condition gave a mixture of
N-7 and N-9 alkylated bases, which were isolated by delicate
column chromatography. Both isomers were determined by NMR
spectroscopic data analysis including HMBC correlation between
protons H2 and H20, and carbons C4 and C5. Bis(POM)-ANP
chloropurine derivatives 7 and 8 were first converted to hypo-
xanthine 9 and guanine 10 derivatives in 86% and 85% yields,
respectively, using diluted HCOOH at 40 ꢀC for 20 h. Compounds
7 and 8 underwent also nucleophilic substitution on the C6-
position with cyclopropylamine to afford 11 and 12 in good
yield. From compound 8, a tricyclic derivative 13 was obtained in
48% yield by treatment with 2-chloroacetaldehyde (50 wt. % in
H2O) in a dioxane/water mixture at 70 ꢀC for 6 h [13]. During our
investigations, we did not observe the degradation of the (POM)
biolabile group proving the efficiency of our strategy in terms of
number of steps and overall yield. However, in order to facilitate
the isolation of the N-9 isomers, substitutions at C6-position
were alternatively proceeded before the Mitsunobu reaction.
The desired C6-substituted-purine bases 14bee and 15cee were
Fig. 1. Some nucleoside phosphonates and target compounds.
5-substituted uracil as an one-step efficient tool to obtain in good
yield a large library of (E)-4-phosphono-but-20-en-10-yl pyrimidine
nucleosides, which some of them exhibit submicromolar activities
against VZV [9,16]. However, it is well-known that nitrogen con-
taining heterocycles can decrease the catalytic metals through
a poisoning mechanism. Usually, metathesis reaction involving
purine nucleosides requires exocyclic amine protection to avoid
catalyst poisoning and often gives very low yields. Thus, to
circumvent this problem, we aimed to determine the most suitable
conditions for an efficient synthesis of (E)-4-phosphono-but-20-en-
10-yl purines in their prodrug form. We thought that selected
purines could be directly introduced through a N-alkylation with
the corresponding activated (E)-4-phosphono-but-20-en-10-yl
counterpart either under SN2 or under Mitsunobu conditions.
Thus, starting with bis(POM)-allyl phosphonate (2), the (E)-
bis(POM)-4-bromo-but-2-en-1-yl phosphonate (30) was obtained
in 73% yields by cross metathesis of bis-(POM)-allyl phosphonate
(2) with (E)-1,4-dibromobut-2-ene. Unfortunately, the N-alkylation
reaction of adenine in the presence of Cs2CO3 [17] and synthon 30 in
anhydrous DMF failed, because of major formation of bis(POM)-
but-1,3-dienyl phosphonate 4 resulting from undesired bromine
elimination (Scheme 1).
We then turned our attention to the introduction of the nucle-
obase under Mitsunobu conditions [18] (Scheme 2). Firstly, the
hitherto unknown (E)-bis(POM)-4-hydroxy-but-2-en-1-yl phos-
phonate 3 bearing hydroxyl was prepared by cross metathesis
reaction with bis(POM) allylphosphonate (2) and 2-buten-1,4-diol
in 74% yields.
Starting from dimethylallylphosphonate, compound 2 was ob-
tained according our previous procedure, using chlor-
omethylpivalate in the presence of sodium iodine during 48 h in
84% yield. (E)-4-Hydroxy-bis(POM)-but-2-enylphosphonate 3, the
key compound of our strategy, was prepared through cross
metathesis reaction between cis-but-2-en-1,4-diol and 2 in the
presence of 5 mol% of Ru catalyst during 16 h in CH2Cl2 at reflux.
obtained from
5 or 6 by nucleophilic substitution with
substituted primary amines and Et3N in nBuOH with yields
ranging from 80 to 92%. Compounds 14aee and 15a,c,d,e,f can be
readily converted in bis(POM) ANP derivatives 16aee and
17a,c,d,e,f by coupling reaction with synthon 3 as described
previously, in 38e52% overall yield. All the structures were
confirmed by 1H, 13C, 31P NMR and HRMS analysis.
The title bis(POM) (E)-4-phosphono-but-20-en-10-yl acyclic
nucleosides 7e13 and 16, 17 were subjected to an in vitro antiviral
screening using a wide spectrum of DNA viruses, in HEL cell
cultures, for vaccinia virus (VV), herpes simplex virus-1(KOS)
(HSV-1), herpes simplex virus-2(G) (HSV-2), varicella-zoster virus
(VZV TKþ and TKꢁ) as summarized in Table 1.
Among the synthesized compounds some of them show potent
inhibitory activities against several DNA viruses, in particular
compounds 10, 12 and 17f, exhibiting submicromolar activities.
Even if the active compounds against DNA virus replication do not
cause a microscopically detectable alteration of cell morphology
(MCC) at concentration up to 100 mM, they were found to be rather
cytostatic. Therefore, it is currently unclear whether the activity
observed for these compounds is due to a specific antiviral effect or
to an antiproliferative activity. It is interesting to notice that the
Scheme 1. Purine base alkylation through the use of halogenated synthon 30.

128
U. Pradère et al. / European Journal of Medicinal Chemistry 57 (2012) 126e133
Scheme 2. Synthesis of (E)-4-hydroxy-bis(POM)-but-2-enylphosphonate.Reagents and conditions (a) NaI, chloromethylpivalate, CH3CN, reflux, 48 h, 84% (b) (Z)-2-buten-1,4-diol,
[Ru] ¼ catalyst (5 mol%), CH2Cl2, reflux, 12 h, 74%.
bis(POM)-ANP derivatives bearing 2-amino 6-substituted purines
(8, 10, 12 and 17a,f) displayed a (sub)micromolar activity against
VZV in contrast with the inactive hypoxanthine analogues (7, 9, 11).
It is tempting to assume that the 2-aminopurine derivatives owe
their antiviral activities upon conversion to the corresponding
guanine derivatives. This assumption is in line with the rather
pronounced cytostatic activity of these compounds, since guanine-
bearing compounds such as PMEG are known to be quite inhibitory
to cell proliferation.
Toro virus in Vero cell cultures and Vesicular stomatitis, Coxsackie
B4 and respiratory syncytial virus in HeLa cell cultures (data not
shown).
3. Conclusion
In summary, a novel series of 4-phosphono-but-2-en-1-yl
purine nucleosides bis(POM) prodrugs, were successfully synthe-
sized by an efficient one step strategy under Mitsunobu conditions
between purines and bis-(POM) (E)-4-phosphono-but-2-enol syn-
thon 3. All synthesized molecules have been evaluated against
several DNA and RNA viruses. Submicromolar activities against HIV
and VZV were displayed for some of these compounds, which
reinforce the choice of our unsaturated side chain for the devel-
opment of antiviral agents. This encouraging result allows us to
prepare new ANPs in their prodrug form bearing a modified
heterocycle moiety in our future drug discovery program.
Among the tested final molecules against Feline herpes virus in
CRFK cell cultures (data not shown) only compound 10 showed
activity against Feline herpes virus at an EC50 of 11
observed cytotoxicity up to 100 M (Table 2).
mM with no
m
All new compounds were also evaluated against HIV-1(IIIB) or
HIV-2(ROD) in CEM cell cultures. Whereas most of them are devoid
of anti-HIV-1 and -2 activity (data not shown), we report in Table 3
the micromolar and submicromolar activities of compounds 17a
and 17f. While compound 17a exhibits micromolar anti-HIV-1 and
anti-HIV-2 activities with an EC50 of 6.6
m
M and 6.3
mM respec-
4. Experimental section
tively, 17f displayed a potent submicromolar anti-HIV-2 activity
with an EC50 of 0.28
was 28.5 (Table 3).
mM. Its selectivity index (ratio CC50(CEM)/EC50
)
4.1. Chemistry
None of the compounds displayed a specific antiviral effect
against a wide panel of other viruses including vesicular-stomatitis
virus, influenza virus type A (H1N1 and H3N2) and B in MDCK cell
cultures, para-influenza-3 virus, reovirus-1, Sindbis virus and Punta
Commercially available chemicals were of reagent grade and
used as received and solvents were dried following standard
procedure. The reactions were monitored by thin layer chroma-
tography (TLC) analysis using silica gel plates (Kieselgel 60F254, E.
Scheme 3. Reagents and conditions (a) (E)-4-hydroxy-bis(POM)-but-2-enylphosphonate 3, PPh3, DIAD, dioxane, r.t., 12 h; (b) H2O/formic acid (1/1), 40 ꢀC, 20 h; (c) cyclopropyl-
amine/CH2Cl2 (1/10), 40 ꢀC; 20 h (d) 1,4-dioxane/H2O (1/1), 2-chloroacetaldehyde (50 wt. % in H2O), 70 ꢀC, 6 h, 48%; (e) appropriate amine derivatives, Et3N, nBuOH, reflux, 16h.

U. Pradère et al. / European Journal of Medicinal Chemistry 57 (2012) 126e133
129
Table 1
Antiviral activity and cytotoxicity of ANP prodrugs against VV, HSV and VZV in HEL cell cultures.
Compounds
EC50a m
M
Cytotoxicity mM
MCCb
(
mM)
CC50 (mM)
c
VV
(Lederle)
HSV-1
(KOS)
HSV-2 (G)
VZV
(TKe
TKþ (OKA)
TKe (07/1)
KOS ACV)
7
8
9
10
11
12
13
16a
17a
16b
16c
17c
16d
17d
16e
17e
17f
>20
>20
>100
100
>100
20
>100
>100
>20
>100
>100
>100
>100
>100
>100
>100
16 ꢂ 4
>250
5
>20
>20
>100
3.9
>20
>20
>20
>20
>100
7.8
>100
7.2
>100
45
20
>100
>100
>100
>100
>100
>100
>100
6.5 ꢂ 2.5
0.4
>20
4.4
>100
1.05 ꢂ 0.2
55.7
>20
2.2
>20
100
10.5
5.4
51.9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
5.8
>100
16.3
>100
20
0.8 ꢂ 0.4
4.1 ꢂ 0.1
>100
16.3
>100
>100
20
>100
>100
>100
>100
>100
>100
>100
6 ꢂ 3
0.4
47.2
39.6
0.8 ꢂ 0
0.8 ꢂ 0
40.7
2.3 ꢂ 0.1
nd
11.6
36 ꢂ 1
>100
37.2
60
11.4
9
32.3
17.3
12.56
6.8 ꢂ 1.2
>100
100
32.4
20
6.34
>20
>20
13.53
12.0 ꢂ 0
>100
>100
43.6
>20
20
>20
>100
0.5 ꢂ 0.3
15.0 ꢂ 1.5
117
20
>100
>100
>100
>100
>100
>100
>100
7.5 ꢂ 4.5
250
100
100
>100
>100
>100
>100
nd
46
0.1 ꢂ 0.4
1.6 ꢂ 0.4
134 ꢂ 10
339
Acyclovir
Brivudine
Cidofovir
2.7
0.08
1.2
250
1
50
1
0.01
nd
10
nd
nd
Bold numbers represents the compounds which possess an antiviral activity <10 mM.
a
50% Effective concentration or compound concentration required to reduce virus-induced cytopathicity by 50%.
Minimum compound concentration that causes a microscopically detectable alteration of cell morphology.
50% Cytostatic concentration or compound concentration required to reduce cell growth by 50%.
b
c
Merck). Column chromatography was performed on Silica Gel 60 M
4.1.2. (E)-Bis(POM)-4-bromo-but-2-en-1-yl phosphonate (30)
(0.040e0.063 mm, E. Merck). The 1H, 31P and 13C NMR spectra were
recorded on a Varian InovaUnity 400 spectrometer (400 MHz) in (d4)
methanol, CDCl3, shift values in parts per million relative to SiMe4
as internal reference. High Resolution Mass spectra were per-
formed by the Mass Spectrometry Center of Blaise Pascal University
(Program Masslynx 4.0) (Aubière, France).
To a dry CH2Cl2 solution (40 mL) of bis-(POM)-allylphosphonate
2 (1 g, 2.82 mmol) and trans-1,4-dibromo-but-2-en (1.21 g,
5.64 mmol) under argon was added RuCl2(PPh3)IMesBenzylidene
Nolan’s catalyst (120 mg, 0.14 mmol, 5%). After 12 h stirring at 40 ꢀC,
volatiles were evaporated and the residue was purified by silica gel
column chromatography (AcOEt/Petroleum ether 1:1) to give pure
(E)-bis-(POM)-4-bromo-but-en-1-yl phosphonate 30 (904 mg,
4.1.1. (E)-Bis(POM)-4-hydroxy-but-2-en-1-yl phosphonate (3)
To a dry CH2Cl2 solution (30 mL) of bis(POM) allylphosphonate
(500 mg, 1.41 mmol), and 2-buten-1,4-diol (604 mg, 2.82 mmol)
under argon was added RuCl2(PPh3)IMesBenzylidene Nolan’s
catalyst (60 mg, 0.071 mmol, 5%). After 48 h stirring at 40 ꢀC,
volatiles were evaporated and residue was purified by silica gel
column chromatography (AcOEt/Petroleum ether 1:1) to give pure
(E)-bis(POM)-4-hydroxy-but-2-en-1-yl phosphonate 3 (402 mg,
2.04 mmol, 73%). 1H NMR (400 MHz, CDCl3)
CH), 5.74e5.62 (m, 5H, HPeCH2eCH
(dd, J ¼ 7.5, 3.5 Hz, 2H, CH2eBr), 2.70 (dd, J ¼ 22.7, 7.3 Hz, 2H, HPeCH2),
1.23 (s, 18H, H C(CH3)3). 13C NMR (100 MHz, CDCl3)
176.3, 132.1,
d
5.93e5.83 (m,1H, HPe
]
CH2eCH
]CH and HOeCH2eO), 3.92
d
131.9, 122.7, 122.6, 81.2, 38.3, 31.2, 30.9, 29.5, 26.5. 31P NMR
(162 MHz, CDCl3)
d
26.77. HRMS (ESI): m/z [M þ Na]þ calcd for
C16H28O7NaBrP: 465.0654, found: 465.0671.
1.06 mmol, 74%). 1H NMR (400 MHz, CDCl3)
CH), 5.73e5.55 (m, 5H, HPeCH2eCH
d
5.88e5.79 (m, 1H, HPe
CH, HOeCH2eO), 4.11
4.1.3. (E)-Bis(POM)-but-1,3-dien-1-yl phosphonate (4)
]
]
To a dry DMF solution (1 mL) of adenine (29 mg, 0.215 mmol)
and cesium carbonate (74 mg, 0.0225 mmol) was added (E)-bis-
(POM)-4-bromo-but-en-1-yl phosphonate 30 (100 mg, 0.225 mmol)
in DMF (0.5 mL). After 24 h stirring at room temperature, all
CH2eCH
(t, J ¼ 7.4 Hz, 2H, HCH2eOH), 2.68 (dd, J ¼ 22.4, 7.3 Hz, 2H, HPeCH2),
2.03 (s, 1H, HOH), 1.22 (s, 18H, HC(CH3)3). 13C NMR (100 MHz,
CDCl3)
d 176.9, 135.9, 135.7, 119.0, 118.9, 81.6, 81.5, 62.9, 38.7, 31.4,
30.1, 26.8. 31P NMR (162 MHz, CDCl3)
d 27.54. HRMS (ESI): m/z
[M þ Na]þ calcd for C16H29NaO8P: 403.1498; found 403.1518.
Table 3
Anti-HIV-1 and HIV-2 activity in CEM cells and cytostatic activity of compounds 17a
and 17f.
Compounds EC50
HIV-1
(
m
M)
IC50
(
m
M)
SIc
a
b
Table 2
HIV-2
L1210
CEM
HeLa
Anti-Feline Herpes Virus activity and cytotoxicity of compound 10 in Crandell-Rees
Feline Kidney cells.
17a
17f
6.6 ꢂ 3.0 6.3 ꢂ 3.2 128 ꢂ 25 46 ꢂ 27 128 ꢂ 25
7.3
28.5
>0.4
0.28 ꢂ
37 ꢂ 6
8.0 ꢂ 2
37 ꢂ 6
a
b
Compound
EC50
(
m
M) Feline
CC50 (mM)
0.014
Herpes Virus
Bold numbers represents the compounds which possess an antiviral activity <10
M.
10
11 ꢂ 0.3
1.2 ꢂ 0.3
>100
>100
μ
m
Ganciclovir
a
50% Effective concentration or compound concentration required to protect
a
50% Effective concentration or compound concentration required to inhibit
virus-induced cytopathicity by 50%.
CEM cells against the cytopathicity (giant cell formation) of HIV by 50%.
b
50% Cytostatic concentration or compound concentration required to reduce
b
50% Cytotoxic concentration or compound concentration required to reduce the
L1210, CEM and HeLa cell proliferation by 50%.
c
viability of CRFK cells by 50%.
Selectivity index; ratio CC50/EC50 in CEM cell culture.

130
U. Pradère et al. / European Journal of Medicinal Chemistry 57 (2012) 126e133
volatiles were evaporated and the residue was purified by silica gel
1.20 (s, 18H, HC(CH3)3). 13C NMR (100 MHz, CDCl3)
153.9, 151.4, 137.2, 130.6, 130.4, 122.7, 122.6, 116.8, 81.7, 81.6, 44.8,
d 176.9, 159.0,
column chromatography (AcOEt/Petroleum ether 1:3) to give by-
product 4 as major compound. 1H NMR (400 MHz, CDCl3)
d
7.10
38.7, 31.4, 30.0, 26.8. 31P NMR (162 MHz, CDCl3)
d 27.04. HRMS
(ddd, J ¼ 22.2, 16.9, 10.6 Hz, 1H, HCH), 6.38 (tdd, J ¼ 16.9, 10.4, 1.8 Hz,
(ESI): m/z [M þ Na]þ calcd for C21H32N5O8NaP: 536.1886; found
1H, HCH), 5.81e5.50 (m, 7H, HCH2, HCH, HOeCH2eO), 1.20 (s, 18H,
536.1890.
H
C(CH3)3). 13C NMR (100 MHz, CDCl3)
d
176.8, 149.7, 149.6, 135.4,
135.1, 126.2, 117.9, 116.0, 81.5, 81.5, 38.7, 26.8. 31P NMR (162 MHz,
CDCl3) 18.86.
4.1.8. N9-(40-Bis(POM)-phosphinyl-20-butenyl)-6-
cyclopropylaminopurine (11)
d
A solution of compound 7 (24 mg, 0.046 mmol) in (1:10)
mixture of cyclopropylamine (0.2 mL) and dichloromethane (2 mL)
was stirred for 20 h at 40 ꢀC. After evaporation of all volatiles, the
residue was purified by silica gel column chromatography (MeOH/
CH2Cl2 4:96) to give compound 11 (20.4 mg, 0.038 mmol, 82%). 1H
4.1.4. N9-(40-Bis(POM)-phosphinyl-but-20-enyl)-6-chloropurine (7)
To a dioxane solution (3 mL) of 3 (100 mg, 0.261 mmol), 6-
chloropurine 5 (49 mg, 0.313 mmol), and triphenylphosphine
(82 mg, 0.313 mmol) under argon at 10 ꢀC was added dropwise di-
isopropylazodicarboxylate (63 mg, 0.313 mmol). After 12 h stirring
at room temperature, volatiles were evaporated, and residue was
purified by silica gel column chromatography (MeOH/CH2Cl2 1:99)
to give compound 7 (97 mg, 0.187 mmol, 71%). 1H NMR (400 MHz,
0
NMR (400 MHz, CDCl3)
d 8.47 (s, 1H, H2), 7.75 (s, 1H, H8), 5.95 (s, 1H,
0
0
HNH), 5.93e5.84 (m, 1H, H2 ), 5.69e5.61 (m, 5H, H3 , HOeCH2eO), 4.78
0
(t, J ¼ 5.1 Hz, 2H, H1 ), 3.04 (d, J ¼ 3.0 Hz, 1H, HNHCH), 2.70 (dd,
0
J ¼ 22.6, 7.3 Hz, 2H, H4 ), 1.21 (s, 18H, HC(CH3)3), 0.94 (td, J ¼ 8.4,
CDCl3)
d
8.73 (s, 1H, H2), 8.14 (s, 1H, H8), 5.94e5.85 (m, 1H, H2 ),
6.9 Hz, 2H, HNHCHCH2), 0.68e0.64 (m, 2H, HNHCHCH2). 13C NMR
0
5.80e5.71 (m, 1H, H3 ), 5.71e5.67 (m, 4H, HOeCH2eO), 4.88 (t,
(100 MHz, CDCl3) d 176.8, 155.8, 153.3, 149.0, 139.5, 130.1, 129.9,
J ¼ 5.2 Hz, 2H, H1 ), 2.71 (dd, J ¼ 22.8, 7.1 Hz, 2H, H4 ), 1.21 (s, 18H,
C(CH3)3). 13C NMR (100 MHz, CDCl3)
176.8, 152.0, 151.6, 151.1,
144.7, 131.6, 128.9, 128.7, 124.6, 124.5, 81.6, 81.5, 45.5, 45.4, 38.7,
123.2, 123.0, 119.8, 81.6, 81.5, 44.8, 38.7, 31.5, 30.1, 26.8, 23.7, 7.4. 31
P
0
0
H
d
NMR (162 MHz, CDCl3)
d
26.57. HRMS (ESI): m/z [M þ H]þ calcd for
C24H37N5O7P: 538.2431; found 538.2430.
31.4, 30.0, 26.8. 31P NMR (162 MHz, CDCl3)
d 26.05; HRMS (ESI): m/z
[M þ Na]þ calcd for C21H30N4NaO7PCl: 539.1438; found 539.1449.
4.1.9. N9-(40-Bis(POM)-phosphinyl-20-butenyl)-2-amino-6-
cyclopropylaminopurine (12)
4.1.5. N9-(40-Bis(POM)-phosphinyl-but-20-enyl)-2 amino-6-
chloropurine (8)
A solution of compound 8 (20 mg, 0.037 mmol) in (1:10)
mixture of cyclopropylamine (0.2 mL) and dichloromethane (2 mL)
was stirred for 20 h at 40 ꢀC. After evaporation of all volatiles, the
residue was purified by silica gel column chromatography (MeOH/
CH2Cl2 3:97) to give compound 12 (16.0 mg, 0.029 mmol, 77%). 1H
0
To a dioxane solution (4.5 mL) of 3 (160 mg, 0.417 mmol), 2-
amino-6-chloropurine 6 (107 mg, 0.627 mmol), and triphenyl-
phosphine (164 mg, 0.627 mmol) under argon at 10 ꢀC was added
dropwise di-isopropylazodicarboxylate (127 mg, 0.627 mmol).
After 12 h stirring at room temperature, volatiles were evaporated,
and residue was purified by silica gel column chromatography
(MeOH/CH2Cl2 2:98) to give compound 8 (112 mg, 0.209 mmol,
NMR (400 MHz, CDCl3)
5.76 (s, 1H, HNH), 5.67e5.57 (m, 5H, H3 , HOeCH2eO), 4.79 (s, 2H,
d
7.44 (s, 1H, H2), 5.89e5.81 (m, 1H, H2 ),
0
0
H
NH2), 4.61 (t, J ¼ 5.1 Hz, 2H, H1 ), 3.05e2.95 (m, 1H, HNHCH), 2.69
0
(dd, J ¼ 23.0, 7.0 Hz, 2H, H4 ), 1.22 (s, 18H, HC(CH3)3), 0.85 (td, J ¼ 6.9,
50%). 1H NMR (400 MHz, CDCl3)
d
7.74 (s, 1H, H8), 5.88e5.79 (m, 1H,
5.4 Hz, 2H, HNHCHCH2), 0.63e0.58 (m, 2H, HNHCHCH2). 13C NMR
0
0
H2 ), 5.72e5.59 (m, 5H, H3 , HOeCH2eO), 5.28 (s, 2H, HNH2), 4.65 (t,
(100 MHz, CDCl3)
d 176.8, 160.1, 156.3, 151.0, 136.9, 130.6, 130.4,
J ¼ 5.1 Hz, 2H, H1 ), 2.70 (dd, J ¼ 22.7, 7.2 Hz, 2H, H4 ), 1.20 (s, 18H,
C(CH3)3). 13C NMR (100 MHz, CDCl3)
176.8, 159.1, 153.6, 151.3,
141.8, 129.4, 129.3, 125.1, 123.6, 123.5, 81.6, 81.5, 44.9, 38.7, 31.4,
122.4,122.3,114.6, 81.6, 44.4, 44.3, 38.7, 31.4, 30.0, 26.8, 23.7, 7.4. 31
P
0
0
H
d
NMR (162 MHz, CDCl3)
d
26.77; HRMS (ESI): m/z [M þ H]þ calcd for
C24H38N6O7P: 553.2531; found 553.2540.
30.0, 26.8. 31P NMR (162 MHz, CDCl3)
d 26.35. HRMS (ESI): m/z
[M þ Na]þ calcd for C21H31N5O7NaPCl: 554.1547, found: 554.1566.
4.1.10. (E)-N3-(40-Bis(POM)-phosphinylbut-20-enyl)-3,9-dihydro-9-
oxo-5H-imidazo[1,2-a]purine (13)
4.1.6. N9-(40-Bis(POM)-phosphinyl-but-20-enyl)hypoxanthine (9)
A solution of 7 (24 mg, 0.046 mmol) in a (1:1) mixture of water
(0.75 mL) and formic acid (0.75 mL) was stirred for 20 h at 40 ꢀC.
After evaporation of all volatiles, the residue was purified by silica
gel column chromatography (MeOH/CH2Cl2 5:95) to give
compound 9 (20 mg, 0.040 mmol, 86%). 1H NMR (400 MHz, CDCl3)
To a 1,4-dioxane/H2O (2 mL, 1/1, v/v) solution of N9-(40-bis(-
POM)-phosphinylbut-20-enyl)-2-amino-6-chloropurine 8 (116 mg,
0.218 mmol) was added 2-chloroacetaldehyde (2.5 mL, 50 wt. % in
H2O). The resulting mixture was stirred 6 h at 70 ꢀC and was
extracted with ethyl acetate. Combined organic layers were dried
over MgSO4 and concentrated in vacuo. Chromatography over silica
gel in CH2Cl2/MeOH 98:2 afforded 56 mg (48%) of desired product
d
12.94 (s, 1H, HNH), 8.17 (s, 1H, H2), 7.81 (s, 1H, H8), 5.94e5.83 (m,
1H, H2 ), 5.78e5.60 (m, 5H, H3 , HOeCH2eO), 4.78 (t, J ¼ 5.2 Hz, 2H,
13 as a white solid. 1H NMR (400 MHz, CDCl3)
7.66 (d, J ¼ 2.8 Hz,1H, H7), 7.64 (s,1H, H2), 7.25 (d, J ¼ 7.9 Hz,1H, H7),
d 11.63 (s, 1H, NH),
0
0
H1 ), 2.72 (dd, J ¼ 22.7, 7.2 Hz, 2H, H4 ), 1.21 (s, 18H, HC(CH3)3). 13
C
0
0
0
0
NMR (100 MHz, CDCl3)
d
176.8, 159.1, 148.9, 145.0, 139.7, 129.7,
5.85 (dq, J ¼ 16.0, 5.6 Hz, 1H, H2 ), 5.70e5.60 (m, 5H, HOeCH2eO, H3 ),
129.5, 124.5, 123.8, 123.7, 81.6, 45.3, 38.7, 31.4, 30.0, 26.8. 31P NMR
4.65 (t, J ¼ 4.8 Hz, 2H, H1 ), 2.72 (dd, J ¼ 22.4, 7.2 Hz, 2H, H4 ), 1.18 (s,
0
0
(162 MHz, CDCl3)
24H26N4O8Na: 521.1648; found 521.1625.
d
26.40. HRMS (ESI): m/z [M þ Na]þ calcd for
18H). 13C NMR (100 MHz, CDCl3)
d
176.92, 152.28, 150.32, 146.12,
C
138.07, 130.32 (d, J ¼ 15 Hz), 122.52 (d, J ¼ 11.6 Hz), 115.91 (d,
J ¼ 18.2 Hz), 107.35, 81.67 (d, J ¼ 6.2 Hz), 38.66, (d, J ¼ 2.1 Hz), 31.28,
4.1.7. N9-(40-Bis(POM)-phosphinyl-but-20-enyl)guanine (10)
29.89, 26.76. 31P NMR (162 MHz, CDCl3)
d 26.81; HRMS (ESI): m/z
A solution of compound 8 (22 mg, 0.041 mmol) in a (1:1)
mixture of water (0.75 mL) and formic acid (0.75 mL) was stirred
for 20 h at 40 ꢀC. After evaporation of all volatiles, the residue was
purified by silica gel column chromatography (MeOH/CH2Cl2 10:90)
to give compound 10 (18.0 mg, 0.035 mmol, 86%). 1H NMR
[M þ H]þ calcd for C23H33N5O8P: 538.20613, found: 538.20578.
4.1.11. General procedure 1: for modification at C-6 position of 6-
chloropurine (5) or 2-amino-6-chloropurine (6)
To a solution of 2-amino-6-chloropurine or 6-chloropurine
(1 eq.) in n-butanol was added substituted amine (3 eq.) and trie-
thylamine (3 eq.). The mixture is stirred during 16 h at reflux then
the solvent is evaporated under reduced pressure. The crude is
(400 MHz, CDCl3)
d 12.11 (s, 1H, HNH), 7.60 (s, 1H, H8), 6.65 (s, 2H,
0
0
HNH2), 5.95e5.84 (m, 1H, H2 ), 5.73e5.62 (m, 5H, H3 , HOeCH2eO),
0
0
4.60 (t, J ¼ 4.7 Hz, 2H, H1 ), 2.72 (dd, J ¼ 22.6, 7.3 Hz, 2H, H4 ),

U. Pradère et al. / European Journal of Medicinal Chemistry 57 (2012) 126e133
131
purified on a silica gel column chromatography with dichloro-
methane and methanol (96/4) as eluant to give pure desired
product 14bee, 15cee.
isopropylaniline, compound 15e was obtained as a white solid
(83%). 1H NMR (400 MHz, CD3OD)
7.79 (s, 1H, H8), 7.69
d
(d, J ¼ 8.5 Hz, 2H, Harom), 7.20 (d, J ¼ 8.5 Hz, 2H, Harom), 2.88 (hept,
J ¼ 6.8 Hz, 1H, HCHisopropyl), 1.24 (d, J ¼ 6.9 Hz, 6H, HCH3isopropyl). 13
C
4.1.11.1. 6-Allylaminopurine (14b). From 6-chloropurine, using
general procedure 1 and allylamine, compound 14b was obtained
NMR (100 MHz, CD3OD) d 156.65, 153.24, 149.72, 144.22, 143.38,
138.38, 127.96, 127.56, 123.63, 123.41, 114.33, 34.20, 23.57, 23.17.
HRMS (ESI): m/z [M þ H]þ calcd for C14H17N6: 269.1515, found
269.1511.
as a yellow solid (84%). 1H NMR (400 MHz, CD3OD)
d 8.23 (s,1H, H2),
8.08 (s, 1H, H8), 6.03 (ddt, J ¼ 21.1, 10.4, 5.3 Hz, 1H, HC2), 5.29 (dd,
J ¼ 17.2, 1.5 Hz, 1H, HC3b), 5.16 (dd, J ¼ 10.3, 1.4 Hz, 1H, HC3a), 4.24 (d,
J ¼ 12.4 Hz, 2H, HC1). 13C NMR (100 MHz, CD3OD)
d
152.28, 151.90,
4.1.12. General procedure 2: for Mitsunobu reaction to obtain
compounds 16aef and 17aef
150.65, 144.22, 136.02, 126.18, 114.80, 41.99. HRMS (ESI): m/z
[M þ H]þ calcd for C8H10N5: 176.0936, found 176.0927.
To a dioxane solution (4.5 mL) of 3 (160 mg, 0.417 mmol), purine
(0.627 mmol), and triphenylphosphine (164 mg, 0.627 mmol)
under argon at 10 ꢀC was added dropwise di-iso-
propylazodicarboxylate (127 mg, 0.627 mmol). After 12 h stirring at
room temperature, volatiles were evaporated, and residue was
purified by silica gel column chromatography (MeOH/CH2Cl2 2:98)
to give pure desired compound.
4.1.11.2. 6-n-Butylaminopurine (14c). From 6-chloropurine, using
general procedure 1 and n-butylamine, compound 14c was ob-
tained as a white solid (91%). 1H NMR (400 MHz, CD3OD)
d 8.51 (s,
1H, H2), 7.99 (s, 1H, H8), 3.38 (dt, J ¼ 8.6, 5.2 Hz, 2H, HC1), 1.55 (tt,
J ¼ 7.6, 5.3 Hz, 2H, HC2), 1.44e1.33 (m, 2H, HC3), 0.98 (t, J ¼ 6.6 Hz,
3H, HC4). 13C NMR (100 MHz, CD3OD)
d 152.54, 151.90, 150.71,
144.22, 124.57, 43.71, 30.87, 20.23, 14.02. HRMS (ESI): m/z [M þ H]þ
4.1.12.1. N9-(40-Bis(POM)-phosphinylbut-20-enyl)adenine
(16a).
calcd for C9H14N5: 192.1249, found 192.1240.
From 6-aminopurine, using general procedure 2, compound 16a
was obtained as a colourless oil (45%). 1H NMR (400 MHz, CDCl3)
0
4.1.11.3. 2-Amino-6-n-butylaminopurine (15c). From 2-amino-6-
d
8.36 (s, 1H, H2), 7.81 (s, 1H, H8), 5.95e5.85 (m, 1H, H2 ), 5.74e5.60
0
0
chloropurine, using general procedure
compound 15c was obtained as a white solid (85%). 1H NMR
(400 MHz, CD3OD)
8.48 (s, 1H, H8), 3.36 (dt, J ¼ 19.4, 7.5 Hz, 2H,
1
and n-butylamine,
(m, 7H, H3 , HOeCH2eO, HNH2), 4.80 (t, J ¼ 5.0 Hz, 2H, H1 ), 2.72 (dd,
J ¼ 22.6, 7.3 Hz, 2H, H4 ), 1.23 (s, 18H, HC(CH3)3). 13C NMR (100 MHz,
0
d
CDCl3) d 176.8, 155.4, 153.1, 149.9, 140.1, 130.0, 129.8, 123.4, 123.2,
H
C1), 1.55 (qt, J ¼ 7.7 Hz, 2H, HC2), 1.45e1.30 (m, 2H, HC3), 0.99 (t,
119.6, 81.6, 44.9, 38.7, 31.5, 30.1, 26.8. 31P NMR (162 MHz, CDCl3)
J ¼ 6.5 Hz, 3H, HC4). 13C NMR (100 MHz, CD3OD)
d
158.84, 154.28,
d
26.50. HRMS (ESI): m/z [M þ H]þ calcd for C21H33N5O7P: 498.2118;
153.25, 144.22, 117.38, 43.71, 30.87, 20.23, 14.02. HRMS (ESI): m/z
found 498.2127.
[M þ H]þ calcd for C9H15N6: 207.1358; found 207.1356.
4.1.12.2. N9-(40-Bis(POM)-phosphinylbut-20-enyl)-2,6-diaminopurine
(17a). From 2,6-diaminopurine, by using general procedure 2,
compound 17a was obtained as a colourless oil (41%). 1H NMR
0
4.1.11.4. 6-Cyclohexylaminopurine (14d). From 6-chloropurine,
using general procedure 1 and cyclohexylamine, compound 14d
was obtained as a white solid (92%). 1H NMR (400 MHz, CD3OD)
(400 MHz, CDCl3)
d 7.50 (s, 1H, H8), 5.94e5.76 (m, 1H, H2 ), 5.71e
0
d
8.50 (s, 1H, H2), 8.00 (s, 1H, H8), 3.28 (qt, J ¼ 7.5 Hz, 1H, HCHcyclo-
hexyl), 1.95 (dt, J ¼ 7.3, 5.7 Hz, 2H, HCH2cyclohexyl), 1.72e1.65 (m, 3H,
CH2cyclohexyl), 1.43 (dt, J ¼ 7.3, 5.8 Hz, 2H, HCH2cyclohexyl), 1.40e1.32
(m, 3H, HCH2cyclohexyl). 13C NMR (100 MHz, CD3OD)
153.82, 152.42,
5.58 (m, 5H, H3 , HOeCH2eO), 5.54 (s, 2H, HNH2), 4.80 (s, 2H, HNH2),
0
0
4.61 (t, J ¼ 5.2 Hz, 2H, H1 ), 2.69 (dd, J ¼ 22.6, 7.2 Hz, 2H, H4 ), 1.21 (s,
18H, HC(CH3)3). 13C NMR (100 MHz, CDCl3)
176.26, 175.83, 164.27,
159.42, 149.75, 140.52, 133.44, 122.34, 121.87, 83.85, 82.95, 46.24,
H
d
d
150.90, 144.22, 124.22, 52.78, 33.42, 33.11, 25.92, 24.92, 24.52.
HRMS (ESI): m/z [M þ H]þ calcd for C11H16N5: 218.1406; found
218.1413.
39.86, 39.47, 32.31, 26.99, 26.78. 31P NMR (162 MHz, CDCl3)
d 26.42;
HRMS (ESI): m/z [M þ H]þ calcd for C21H34N6O7P: 513.2227; found
513.2225.
4.1.11.5. 2-Amino-6-cyclohexylaminopurine (15d). From 2-amino-6-
chloropurine, using general procedure 1 and cyclohexylamine,
compound 15d was obtained as a white solid (92%). 1H NMR
4 .1.12 . 3 . N 9 - ( 4 0- B i s ( P O M ) - p h o s p h inyl b u t - 2 0- e nyl ) - 6 -
allylaminopurine (16b). From 14b, using general procedure 2,
compound 16b was obtained as a colourless oil (52%). 1H NMR
(400 MHz, CD3OD)
d
8.49 (s, 1H, H8), 3.50 (qt, J ¼ 7.0 Hz, 1H,
CHcyclohexyl), 1.88e1.80 (m, 2H, HCH2cyclohexyl), 1.70e1.62 (m, 3H,
CH2cyclohexyl), 1.47e1.40 (m, 2H, HCH2cyclohexyl), 1.38e1.30 (m, 3H,
CH2cyclohexyl). 13C NMR (100 MHz, CD3OD)
158.56, 154.58, 144.22,
(400 MHz, CDCl3)
d
8.39 (s, 1H, H2), 7.75 (s, 1H, H8), 6.10e5.76
0
0
H
H
H
(m, 1H, H2 ), 5.75e5.54 (m, 5H, H3 , HOeCH2eO), 5.25 (ddd, J ¼ 13.7,
0
11.6, 1.4 Hz, 2H, HC3), 4.78 (t, J ¼ 5.0 Hz, 2H, H1 ), 4.33 (d, J ¼ 11.6 Hz,
0
d
2H, HC1), 2.71 (dd, J ¼ 22.6, 7.2 Hz, 2H, H4 ),1.21 (s,18H, HC(CH3)3). 13
C
115.76, 52.78, 33.42, 33.11, 25.92, 24.92, 24.52. HRMS (ESI): m/z
NMR (100 MHz, CDCl3) d 176.26, 175.83, 158.08, 152.42, 151.06,
[M þ H]þ calcd for C11H17N6: 233.1515, found 233.1517.
141.07, 136.02, 133.44, 126.22, 121.87, 114.80, 83.85, 82.95, 46.24,
41.99, 39.86, 39.47, 32.31, 26.99, 26.78. 31P NMR (162 MHz, CDCl3)
4.1.11.6. 6-(4-Isopropyl)phenylaminopurine
(14e). From
6-
d
26.57; HRMS (ESI): m/z [M þ H]þ calcd for C24H37N5O7P:
chloropurine, using general procedure 1 and 4-isopropylaniline,
538.2431; found 538.2440 calculated.
compound 14e was obtained as a white solid (88%). 1H NMR
(400 MHz, CD3OD)
d
8.49 (s, 1H, H2), 8.06 (s, 1H, H8), 7.21 (d,
4 .1.12 . 4 . N 9 - ( 4 0- B i s ( P O M ) - p h o s p h inyl b u t - 2 0- e nyl ) - 6 -
butylaminopurine (16c). From 14c, by using general procedure 2,
compound 16c was obtained as a colourless oil (49%). 1H NMR
J ¼ 7.5 Hz, 2H, Harom), 6.94e6.86 (m, 2H, Harom), 3.04 (hept,
J ¼ 6.5 Hz, 1H, HCHisopropyl), 1.33 (d, J ¼ 6.4 Hz, 6H, HCH3isopropyl). 13
C
NMR (100 MHz, CD3OD)
d
153.24, 149.26, 148.16, 144.22, 143.38,
(400 MHz, CDCl3)
d
8.32 (s, 1H, H2), 7.73 (s, 1H, H8), 5.85e5.93
0
0
138.38, 127.96, 127.56, 124.31, 123.63, 123.41, 34.20, 23.57, 23.17.
HRMS (ESI): m/z [M þ H]þ calcd for C14H16N5: 254.1406, found
254.1406.
(m, 1H, H3 ), 5.71e5.58 (m, 5H, H2 HOeCH2eO), 4.85 (d,
0
0
J ¼ 5.3 Hz, 1H, H1 a), 4.74 (d, J ¼ 5.4 Hz, 1H, H1 b), 3.44 (t, J ¼ 4.9 Hz,
1H, HC1a), 3.35 (t, J ¼ 4.9 Hz, 1H, HC1b), 2.66 (dd, J ¼ 11.9, 5.4 Hz,
0
2H, H4 ), 2.01 (s, 1H, HNH), 1.58 (ddt, J ¼ 12.8, 7.6, 5.0 Hz, 2H, HC2),
4.1.11.7. 2-Amino-6-(4-isopropyl)phenylaminopurine
(15e).
1.46e1.36 (m, 2H, HC3), 1.28 (s, 18H, HC(CH3)3), 0.99 (t, J ¼ 6.6 Hz,
From 2-amino-6-chloropurine, by using general procedure 1 and 4-
3H, HC4). 13C NMR (100 MHz, CDCl3)
d 176.04, 157.21, 152.47,

132
U. Pradère et al. / European Journal of Medicinal Chemistry 57 (2012) 126e133
0
0
151.00, 141.06, 133.43, 125.05, 121.87, 83.40, 46.23, 43.71, 39.66,
32.65, 31.97, 30.87, 26.88, 20.22, 14.01. 31P NMR (162 MHz, CDCl3)
d
H
arom), 6.93 (d, J ¼ 7.5 Hz, 2H, Harom), 5.71e5.58 (m, 6H, H2 , H3 , HOe
0
0
CH2eO), 4.91 (d, J ¼ 5.2 Hz,1H, H1 a), 4.49 (d, J ¼ 5.4 Hz,1H, H1 b), 3.89
26.48. HRMS (ESI): m/z [M þ H]þ calcd for C25H41N5O7P:
(s,1H, HNH), 3.04 (hept, J ¼ 6.3 Hz,1H, HCHisopropyl), 2.66 (dd, J ¼ 11.9,
0
554.2744; found 554.2739.
5.4 Hz, 2H, H4 ), 2.11 (s, 2H, HNH2), 1.33 (d, J ¼ 6.4 Hz, 6H, HCH3iso-
propyl), 1.30 (s, 18H, HC(CH3)3). 13C NMR (100 MHz, CDCl3)
d 176.04,
4.1.12.5. N9-(40-Bis(POM)-phosphinylbut-20-enyl)-2-amino-6-
butylaminopurine (17c). From 15c, using general procedure 2,
compound 17c was obtained as a colourless oil (49%). 1H NMR
161.65, 155.21, 150.38, 143.37, 141.06, 138.38, 133.43, 127.76, 123.52,
122.63, 121.87, 83.40, 46.23, 39.66, 34.19, 32.65, 31.97, 26.88, 23.37.
31P NMR (162 MHz, CDCl3)
for C30H44N6O7P: 631.6748; found 631.6754.
d
26.42; HRMS (ESI): m/z [M þ H]þ calcd
0
0
(400 MHz, CDCl3)
d
8.16 (s, 1H, H8), 5.77e5.63 (m, 6H, H2 , H3 , HOe
0
CH2eO), 4.78 (dd, J ¼ 9.2, 5.5 Hz, 2H, H1 ), 3.44 (t, J ¼ 5.0 Hz,1H, HC1a),
4.1.12.10. N9-(40-Bis(POM)-phosphinylbut-20-enyl)-2-amino-6-
methoxypurine (17f). From 2-amino-6-methyoxypurine, by using
general procedure 2, compound 17f was obtained as a colourless oil
0
3.35 (t, J ¼ 4.9 Hz, 1H, HC1b), 2.66 (dd, J ¼ 11.9, 5.7 Hz, 2H, H4 ), 1.99
(s, 1H, HNH), 1.58 (ddt, J ¼ 12.8, 7.6, 5.0 Hz, 2H, HC2), 1.44e1.35 (m,
2H, HC3), 1.28 (s, 18H, HC(CH3)3), 0.99 (t, J ¼ 6.6 Hz, 3H, HC4). 13C NMR
(100 MHz, CDCl3)
d
176.04, 161.23, 159.32, 150.89, 141.06, 133.43,
(51%). 1H NMR (400 MHz, CDCl3)
d
8.04 (s, 1H, H8), 5.73e5.61
0
0
0
122.90, 121.87, 83.40, 46.23, 43.71, 39.66, 32.65, 31.97, 30.87, 26.88,
(m, 6H, H2 , H3 , HOeCH2eO), 4.76 (d, J ¼ 5.3 Hz, 1H, H1 a), 4.56 (d,
20.22, 14.01. 31P NMR (162 MHz, CDCl3)
d
26.50; HRMS (ESI): m/z
J ¼ 5.4 Hz, 1H, H1 b), 3.99 (s, 3H, HOMe), 3.02 (s, 2H, HNH2), 2.66 (dd,
0
[M þ H]þ calcd for C25H42N6O7P: 569.6170; found 569.6164.
J ¼ 11.9, 5.4 Hz, 2H, H4 ), 1.26 (s, 18H, HC(CH3)3). 13C NMR (100 MHz,
0
CDCl3) d 176.04,164.04,160.79,149.49,137.51, 133.43, 127.28,121.87,
4 .1.12 . 6 . N 9 - ( 4 0- B i s ( P O M ) - p h o s p h i nyl b u t - 20- e nyl ) - 6 -
cyclohexylaminopurine (16d). From 14d, by using general proce-
dure 2, compound 16d was obtained as a colourless oil (42%). 1H
83.40, 53.95, 46.23, 39.66, 32.65, 31.97, 26.88. 31P NMR (162 MHz,
CDCl3)
d
26.51; HRMS (ESI): m/z [M þ H]þ calcd for C22H35N5O8P:
528.2223; found 528.2228.
NMR (400 MHz, CDCl3)
d
8.39 (s, 1H, H2), 8.14 (s, 1H, H8), 5.73e
0
0
0
5.59 (m, 6H, H2 , H3 , HOeCH2eO), 4.97 (d, J ¼ 5.4 Hz, 1H, H1 a),
4.2. Antiviral activity assays
0
4.48 (d, J ¼ 5.4 Hz, 1H, H1 b), 3.78 (tt, J ¼ 7.9, 3.8 Hz, 1H, HCHcy-
0
clohexyl), 2.66 (dd, J ¼ 11.9, 5.4 Hz, 2H, H4 ), 1.92 (dqd, J ¼ 11.4, 5.7,
The antiviral assays, other than the anti-HIV assays, were based
on inhibition of virus-induced cytopathicity or plaque formation in
HEL [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccinia
virus, vesicular stomatitis virus, cytomegalovirus (HCMV), and
varicella-zoster virus (VZV)], Vero (parainfluenza-3, reovirus-1,
Sindbis virus and Coxsackie B4), HeLa (vesicular stomatitis virus,
Coxsackie virus B4, and respiratory syncytial virus) or MDCK
[influenza A (H1N1; H3N2) and influenza B] cell cultures. Confluent
cell cultures (or nearly confluent for MDCK cells) in microtiter 96-
well plates were inoculated with 100 CCID50 of virus (1 CCID50
being the virus dose to infect 50% of the cell cultures) or with 20
plaque forming units (PFU) (for VZV) in the presence of varying
2.1 Hz, 2H, HCH2cyclohexyl), 1.78 (s, 1H, HNH), 1.72 (tdd, J ¼ 11.5, 5.7,
2.2 Hz, 2H, HCH2cyclohexyl), 1.67e1.52 (m, 6H, HCH2cyclohexyl), 1.28 (s,
18H, HC(CH3)3). 13C NMR (100 MHz, CDCl3)
d 176.04, 157.04, 151.05,
150.21, 141.06, 133.43, 124.13, 121.87, 83.40, 52.78, 46.23, 39.66,
33.31, 32.65, 31.97, 26.88, 25.91, 24.72. 31P NMR (162 MHz, CDCl3)
d
26.54; HRMS (ESI): m/z [M þ H]þ calcd for C27H43N5O7P:
580.6417; found 580.6410.
4.1.12.7. N9-(40-Bis(POM)-phosphinylbut-20-enyl)-2-amino-6-
cyclohexylaminopurine (17d). From 15d, using general procedure 2,
compound 17d was obtained as a colourless oil (50%). 1H NMR
0
0
(400 MHz, CDCl3)
d
7.81 (s, 1H, H8), 5.73e5.60 (m, 6H, H2 , H3 , HOe
concentrations (100, 20,. mM) of the test compounds. Viral cyto-
0
0
CH2eO), 4.92 (d, J ¼ 5.3 Hz, 1H, H1 a), 4.51 (d, J ¼ 5.3 Hz, 1H, H1 b),
pathicity was recorded as soon as it reached completion in the
control virus-infected cell cultures that were not treated with the
test compounds. Antiviral activity was expressed as the EC50 or
compound concentration required to reduce virus-induced cyto-
pathogenicity or viral plaque (VZV) plaque formation by 50%. The
minimal cytotoxic concentration (MCC) of the compounds was
defined as the compound concentration that caused a microscopi-
cally visible alteration of cell morphology. Alternatively, the cyto-
static activity of the test compounds was measured based on
inhibition of cell growth. HEL cells were seeded at a rate of
5 ꢃ 103 cells/well into 96-well microtiter plates and allowed to
proliferate for 24 h. Then, medium containing different concen-
trations of the test compounds was added. After 3 days of incuba-
tion at 37 ꢀC, the cell number was determined with a Coulter
counter. The cytostatic concentration was calculated as the CC50, or
the compound concentration required to reduce cell proliferation
by 50% relative to the number of cells in the untreated controls. The
methodology of the anti-HIV assays was as follows: human CEM
(w3 ꢃ 105 cells/cm3) cells were infected with 100 CCID50 of
0
3.71e3.62 (m, 1H, HCHcyclohexyl), 2.66 (dd, J ¼ 11.9, 5.4 Hz, 2H, H4 ),
2.14e2.03 (m, 3H, HNH and HCH2cyclohexyl), 1.75e1.56 (m, 8H,
H
d
CH2cyclohexyl), 1.30 (s, 18H, HC(CH3)3). 13C NMR (100 MHz, CDCl3)
176.04, 159.80, 159.56, 151.27, 141.06, 133.43, 122.02, 121.69, 83.40,
52.78, 46.23, 39.66, 33.31, 32.65, 31.97, 26.88, 25.91, 24.72. 31P NMR
(162 MHz, CDCl3)
d
26.51; HRMS (ESI): m/z [M þ H]þ calcd for
C27H44N6O7P: 595.6333; found 595.6322.
4.1.12.8. N9-(40-Bis(POM)-phosphinylbut-20-enyl)-6-(4-isopropyl)
phenylaminopurine (16e). From 14e, using general procedure 2,
compound 16e was obtained as a colourless oil (39%). 1H NMR
(400 MHz, CDCl3)
d 8.67 (s, 1H, H2), 7.87 (s, 1H, H8), 7.72 (d,
J ¼ 7.5 Hz, 2H, Harom), 7.22 (d, J ¼ 7.5 Hz, 2H, Harom), 5.72e5.59 (m,
0
0
0
6H, H2 , H3 , HOeCH2eO), 4.96 (d, J ¼ 5.4 Hz, 1H, H1 a), 4.48 (d,
0
J ¼ 5.2 Hz, 1H, H1 b), 3.86 (s, 1H, HNH), 2.99 (hept, J ¼ 6.4 Hz, 1H,
0
H
CHisopropyl), 2.66 (dd, J ¼ 11.9, 5.4 Hz, 2H, H4 ), 1.34 (d, J ¼ 6.4 Hz,
6H, HCH3isopropyl), 1.30 (s, 18H, HC(CH3)3). 13C NMR (100 MHz,
CDCl3) 176.04, 153.12, 151.64, 143.37, 141.06, 138.38, 133.43,
127.76, 123.52, 123.04, 121.87, 83.40, 46.23, 39.66, 34.19, 32.65,
d
HIV(IIIB) or HIV-2(ROD)/ml and seeded in 200 mL wells of a micro-
31.97, 26.88, 23.37. 31P NMR (162 MHz, CDCl3)
d
26.48; HRMS
titer plate containing appropriate dilutions of the test compounds.
After 4 days of incubation at 37 ꢀC, HIV-induced CEM giant cell
formation was examined microscopically.
(ESI): m/z [M þ H]þ calcd for C30H43N5O7P: 616.2900; found
616.2909.
4.1.12.9. N9-(40-Bis(POM)-phosphinylbut-20-enyl)-2-amino-6-(4-
isopropyl)phenylaminopurine (17e). From 15e, by using general
procedure 2, compound 17e was obtained as a colourless oil (45%).
Acknowledgement
O.S. is grateful to the French Ministère de l’Enseignement
Supérieur MNERST for a Ph.D. scholarship. The research was
1H NMR (400 MHz, CDCl3)
d
7.88 (s, 1H, H2), 7.22 (d, J ¼ 7.5 Hz, 2H,

U. Pradère et al. / European Journal of Medicinal Chemistry 57 (2012) 126e133
133
P. Meyer, J. Balzarini, L.A. Agrofoglio, Novel antiviral C5-substituted pyrimi-
dine acyclic nucleoside phosphonate selected as human thymidylate kinase
substrates, J. Med. Chem. 54 (2011) 222e232.
supported by the KU Leuven (GOA 10/014). The authors thank L.
Persoons, F. De Meyer, L. Ingels, A. Camps, L. Van den Heurck and S.
Carmans for dedicated technical assistance for the antiviral assays.
[10] A. Montagu, U. Pradére, V. Roy, S.P. Nolan, L.A. Agrofoglio, Expeditious
convergent procedure for the preparation of bis(POC) prodrugs of (E)-
4-phosphono-but-2-en-1-yl nucleoside, Tetrahedron 67 (2001) 5319e5328.
[11] E. De Clercq, G. Andrei, R. Snoeck, L. De Bolle, L. Naesens, B. Degrève,
J. Balzarini, Y. Zhang, D. Schols, P. Leyssen, C. Ying, J. Neyts, Acyclic/carbocyclic
guanosine analogues as anti-herpesvirus agents, Nucleos. Nucleot. Nucleic
Acids 20 (2001) 271e285.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.08.042.
[12] J. Boryski, B. Golankiewicz, E. De Clercq, Synthesis and antiviral activity of
3-substituted derivatives of 3,9-dihydro-9-oxo-5H-imidazo,[1,2-a]purines,
tricyclic analogues of acyclovir and ganciclovir, J. Med. Chem. 34 (1991)
2380e2382.
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